Transforming Growth Factor β Controls CCN3 Expression in Nucleus Pulposus Cells of the Intervertebral Disc

Objective. To investigate transforming growth factor beta (TGF beta) regulation of CCN3 expression in cells of the nucleus pulposus. Methods. Real-time reverse transcription-polymerase chain reaction and Western blot analyses were used to measure CCN3 expression in the nucleus pulposus. Transfections were used to measure the effect of Smad3, MAPKs, and activator protein 1 (AP-1) on TGF beta-mediated CCN3 promoter activity. Lentiviral knockdown of Smad3 was performed to assess the role of Smad3 in CCN3 expression. Results. CCN3 was expressed in embryonic and adult intervertebral discs. TGF beta decreased the expression of CCN3 and suppressed its promoter activity in nucleus pulposus cells. DN-Smad3, Smad3 small interfering RNA, or DN-AP-1 had little effect on TGF beta suppression of CCN3 promoter activity. However, p38 and ERK inhibitors blocked suppression of CCN3 by TGF beta, suggesting involvement of these signaling pathways in the regulation of CCN3. Interestingly, overexpression of Smad3 in the absence of TGF beta increased CCN3 promoter activity. We validated the role of Smad3 in controlling CCN3 expression in Smad3-null mice and in nucleus pulposus cells transduced with lentiviral short hairpin Smad3. In terms of function, treatment with recombinant CCN3 showed a dose-dependent decrease in the proliferation of nucleus pulposus cells. Moreover, CCN3-treated cells showed a decrease in aggrecan, versican, CCN2, and type I collagen expression. Conclusion. The opposing effect of TGF beta on CCN2 and CCN3 expression and the suppression of CCN2 by CCN3 in nucleus pulposus cells further the paradigm that these CCN proteins form an interacting triad, which is possibly important in maintaining extracellular matrix homeostasis and cell numbers.